Treatment of virus infections with ganglionic blocking agents

ABSTRACT

A method for preventing or treating infection or disease in a mammal caused by a virus, such as herpes simplex virus type 1 and 2. A ganglionic blocking agent, such as tetraethylammonium ion or hexamethonium ions, is administered to the mammal in an effective dosage.

This is a continuation of application Ser. No. 08/396,901 filed Mar. 1,1995, now abandoned, which is a continuation of Ser. No. 08/168,409filed Dec. 17, 1993, now abandoned, which is a continuation applicationof Ser. No. 08/043,599 filed Apr. 5, 1993, now abandoned, which is acontinuation of application Ser. No. 07/964,475 filed Oct. 21, 1992, nowabandoned, which is a continuation of application Ser. No. 07/837,696filed Feb. 19, 1992, now abandoned, which is a continuation ofapplication Ser. No. 07/618,514 filed Nov. 21, 1990, now abandoned,which is a continuation of application Ser. No. 07/437,806 filed Nov.17, 1989, now abandoned, which is a continuation of application Ser. No.07/214,881 filed Jul. 5, 1988, patented, now U.S. Pat. No. 4,898,888which is a continuation of application Ser. No. 07/019,116 filed Feb.26, 1987, now abandoned, which is a continuation of application Ser. No.06/756,653 filed Jul. 19, 1985, abandoned, which is acontinuation-in-part of Ser. No. 06/743,889 filed Jun. 12, 1985, nowabandoned, which is a continuation application of Ser. No. 06/631,645filed Jul. 16, 1984, now abandoned which is a continuation-in-part ofapplication Ser. No. 06/456,732 filed Jan. 10, 1983, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a method of preventing or treatinginfection and disease in a mammal caused by a virus. More specifically,the invention relates to the administration of a ganglionic blockingagent to a mammal either before or after the virus has infected thehost.

Viruses are associated with a large number of infections and diseases inmammals, such as man. Although modern medical science has developed sometreatment techniques that are effective to prevent a particular disease,i.e., the polio vaccine, the art generally lacks a method by which alarge number of different virus infections can be effectively preventedor treated.

Past treatments of various diseases caused by viruses have been largelyineffective. To inhibit viral replication, an agent must selectivelyinhibit one or more of the following specific functions: (1) adsorption,(2) uncoating, (3) transcription, (4) protein synthesis, (5) nucleicacid replication, (6) maturation, and (7) release.

For example, various agents developed in an attempt to treat HerpesSimplex Virus type 1 (HSV-1) and Type 2 (HSV-2), (e.g. idoxuridine,cytosine arabinoside, adenine arabinoside, triflurothymidine, andacyclovir) all interfere with viral and host cellular functions. Becauseof host cell toxicity, these agents have been of very limitedeffectiveness for use, especially systemic, in humans to treat orprevent HSV-1 and HSV-2.

Accordingly, there is a strong need for a method that can effectivelytreat or prevent in a mammal infections and diseases caused by viruses,of which the above-mentioned HSV-1 and HSV-2 are merely examples.Although ganglionic blockers have been known in the art, the art hasfailed to recognize that ganglionic blockers can effectively treat andprevent in mammals infections and diseases associated with a variety ofviruses, including HSV-1 and HSV-2.

Various investigators first described the nicotine paralyzing actions ofthe tetraethylammonium (TEA) ion on ganglia as early as 1913 and otherinvestigators reported certain additional properties. Nevertheless, theart largely overlooked the TEA ion until Acheson and Moe in 1945 and1946 and Acheson and Pereira in 1946 published their definitive analysesof the effects of the TEA ion on the cardiovascular system and autonomicganglia. The pharmacological effects of the TEA ion were then studied inman and applied clinically in a variety of disorders in which reversiblesympathetic ganglionic blockade was desired. The subsequent discovery ofthe ganglionic blocking agent, hexamethonium (HM) ion, however,relegated the TEA ion to a minor status as a diagnostic tool andtherapeutic agent.

Various bis-quaternary ammonium salts that were ganglionic blockingagents, such as hexamethonium bromide (HMB), were then developed andstudied independently by Barlow and Ing in 1948 and by Paton and Zaimasin 1949. Marked ganglionic blockage was found when the bridge betweenthe two nitrogen atoms has 5 or 6 methylene groups and markedneuromuscular blockage was found when the bridge consisted of 10 to 12methylene groups.

Various triethylsulfonium salts, such as the monoquaternary andbis-quaternary ammonium ions, also possess ganglionic blocking actions.the synthesis of trimethaphorsulfonate, a ganglionic blocker, occurredin 1949.

Althoughtetraethylammonium chloride (TEAC), a ganglionic blocker, wasbriefly advocated for use in the 1950s to treat the pain associated withherpes zoser, this recommendation was subsequently abandoned. Thus,there was no recognition in the art that TEAC could be used to treatinfections and diseases caused by a variety of other viruses.

The synthesis of secondary amines with ganglionic blocking activityrepresented somewhat of a departure in the chemistry of these blockingagents. The pharmacological properties of mecamylamine hydrochloridewere first reported in the mid-1950's, and the drug was soon thereafterintroduced into therapy. Pempidine was introduced shortly thereafter.

Notwithstanding the long period during which ganglionic blockers havebeen known and studied, the principal therapeutic use of ganglionicblockers, other than in the present invention, is in the treatment ofsevere hypertension and hypertensive crisis in humans. However, newerpotent agents, such as nitroprusside and diazoxide, have virtuallyreplaced the ganglionic blockers for these uses. Hence, until thepresent invention, the art has failed to recognize the ability ofganglionic blocking agents to treat or prevent infections and diseasesin a mammal caused by a virus.

SUMMARY OF THE INVENTION

Quite surprisingly, the present inventor has discovered a method thateffectively treats or prevents infections and diseases in mammals causedby a number of viruses. More particularly, the present inventionprovides a method for preventing or treating infection and diseases in amammal caused by a virus comprising the step of administering to themammal a ganglionic blocking agent in an effective dosage. However, whenthe virus is herpes zoster, the ganglionic blocking agent is nottetraethylammonium chloride (TEAC).

The present invention also provides a method of treating a mammalcomprising the step of administering to the mammal a ganglionic blockingagent in a dosage effective to produce an anti-viral effect with respectto a virus. In this method, when the virus is herpes zoster, theganglionic blocking agent is not TEAC.

In another embodiment, the present invention provides a method oftreating a mammal comprising the step of administering to the mammal aganglionic blocking agent in a dosage effective to inhibit the viralfunction of a virus. Similarly, when the virus is herpes zoster, theganglionic blocking agent is not TEAC.

In the present invention, the ganglionic blocking agent is preferablyselected from tetraethylammonium ion, hexamethonium ion, pentoliniumion, chlorisondamine ion, trimethidinium ion, trimethaphan ion,mecamylamine, pempidine, and homologs thereof. Preferably, the virus isan adenovirus, an arbovirus, an arena virus, a bacteriophage, acoliphage, a coronavirus, a hepatitis virus, a herpetovirus, anoncogenic virus, an orthomyxovirus, a papovavirus, a paramyxovirus, aparvovirus, a picornavirus, a plant virus, a pox virus, a reovirus, aretrovirus, a rhabdovirus, and a togavirus.

The present invention overcomes some of the disadvantages commonlyassociated with previous methods of treating or preventing virusinfections and diseases and obtains the various advantages of theinvention. By administering a ganglionic blocking agent to a mammalinfected by a virus, the present method treats or prevents infectionsand diseases caused by the virus. Moreover, the ganglionic blockingagents, when administered to the infected mammal, produce an anti-viraleffect or inhibit the viral function of the virus.

These and other features and advantages of the present invention will bemade more apparent from the following description of the preferredembodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for preventing or treatinginfection and disease in a mammal caused by a virus. In the method, aganglionic blocking agent is administered to a mammal in an effectivedosage. However, when the virus is herpes zoster, the ganglionicblocking agent is not TEAC.

In general, ganglionic blocking agents or ganglionic blockers are drugsthat block transmission in autonomic ganglia without producing anypreceding or concomitant change in the membrane potentials of theganglion cells. Ganglionic blockers also do not modify the conduction ofimpulses in the preganglionic or postganglionic neurons and they do notprevent the release of acetylcholine (ACh) by preganglionic impulses.Ganglionic blockers produce ganglionic blockade by occupying receptorsites on the ganglion cells and stabilizing the postsynaptic membranesagainst the actions of ACh liberated from the presynaptic nerve endings.

Various secondary effects are also related to the ganglionic blockingfunction. These effects includes the lowering of blood pressure topronounced vasodilator action, mydriasis (pupil dilation), cycloplegia,which may cause temporary blurred vision, ptosis and similar impairmentof physical responses that are generally associated with nerve impulseblocking action.

Various ganglionic blocking agents known in the art can be useddepending upon the virus and the mammal infected by the virus. Thepreferred ganglionic blocking agents are tetraethyl ammonium ion,hexamethonium ion, pentolinium ion, chlorisondamine ion, trimethidiniumion, trimethaphan ion, mecamylamine, pempidine, and homologs thereof. Anappropriate anion, such as chloride or bromide, is present with theganglionic blocking agent in the pharmaceutical preparation. Thesepreferred ganglionic blocking agents have the following chemicalformulas:

                  TABLE 1                                                         ______________________________________                                         ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                     ______________________________________                                    

However, as noted, other ganglionic blockers known in the art can alsobe used.

The diversity of compounds sharing a ganglionic blocking action onautonomic ganglia is illustrated in Table 1 above. The chemicalconfigurations include simple monoquaternary ammonium compounds such astetraethylammonium chloride (TEAC); complex monoquaternary ammoniumcompounds such as phenacylhomatropinium; bis-quaternary ammoniumcompounds such as pentamethonium chloride, hexamethonium chloride, andpentolinium tartrate; asymmetrical diquaternary ammonium compounds suchas trimethidinium methasulfate and chlorisondamine chloride; complexmonosulfonium compounds such as trimethaphan camphorsulfonate; andsecondary amines such as mecamylamine hydrochloride and pempidinehydrochloride. The ganglionic blockers can be made by numerous methodsknown in the art. For example, U.S. Pat. No. 2,653,156 to Deutsch et al.disclosed how to prepare TEAC.

The present method can be used to treat or prevent infection and diseasein a mammal caused by any virus by administering to the mammal aneffective dosage of a ganglionic blocking agent that is effectiveagainst that particular virus. Once taught the present invention, oneskilled in the art can readily select the appropriate ganglionicblocking agent for a particular virus without undue experimentation.However, when the virus is herpes zoster, the selected ganglionicblocking agent is not TEAC.

Preferably, the present method is used to treat or prevent infectionsand diseases caused by a virus selected from the group consisting ofadenoviruses, arboviruses, arena viruses, bacteriophages, bunya viruses,coliphages, coronaviruses, hepatitis viruses, herpetoviruses, oncogenicviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, parvoviruses,picornaviruses, plant viruses, pox viruses, reoviruses, retroviruses,rhabdoviruses, and togaviruses. Examples of these viruses are listed inTable 2. This list, however, is not meant to be inclusive. Hence,infections and diseases in mammals caused by other viruses that can betreated or prevented by administering a ganglionic blocking agent to themammal are within the scope of the present invention.

                  TABLE 2                                                         ______________________________________                                        Virus Family     Examples of Virus                                            ______________________________________                                        Adenovirus                                                                    Arbovirus                                                                     Arenavirus                                                                    Bacteriophages   T2, T4, T6                                                   Bunyavirus                                                                    Coliphages       R17, T3, T5, T7                                              Coronavirus                                                                   Hepatitis        Hepatitis A, B, C                                            Herpetovirus     Herpes Simplex 1,                                                             Herpes Simplex 2,                                                             Cytomegalovirus                                                               Epstein-Barr,                                                                 Varicella                                                    Oncogenicvirus   Retrovirus                                                   Orthomyxovirus   Influenza A, B, C                                                             Parainfluenza                                                Papovavirus      Polyoma                                                                       Papilloma                                                                     SV 40                                                        Paramyxovirus    Newcastle disease                                                             Parainfluenza                                                                 Subacute schlerosing                                                          panencephalitis                                                               Rubeola                                                                       Mumps                                                                         Sendai                                                                        Morbillivirus                                                                 Pneumovirus                                                                   Measles                                                                       Canine Distemper                                             Parvovirus       Adeno assoc.                                                                  Oliphage                                                     Picornavirus     Enterovirus                                                                   Polio                                                                         Coxsackievirus                                                                Echovirus                                                                     Enterovirus                                                                   Rhinovirus                                                                    Hoof & Mouth Disease                                         Plant Virus      Tobacco Mosaic                                                                Turnip Yellow                                                                 Rice Dwarf                                                                    Beet YeIlow                                                  Pox Virus        Variola                                                                       Vaccinia                                                                      Contagious Dermatitis                                                         Cow Pox                                                                       Molluscum Contagiosum                                                         Paravaccinia                                                                  Tana Pox                                                     Reovirus         Orbivirus                                                                     Rotavirus                                                                     Cytoplasmic Polyhedrosis                                     Retrovirus       Sarcoma                                                                       Leukemia                                                                      Lymphoma                                                                      Carcinoma                                                    Rhabdovirus      Rabies                                                                        Marburg                                                      Togavirus        Encephalitis                                                                  Alphavirus                                                                    Flavivirus                                                                    Rubivirus                                                                     Rubellavirus                                                                  YellowFever                                                  ______________________________________                                    

Descriptions and structures of many of the viruses listed in Table 2 areprovided in Davis et al., Microbiology, Including Immunology & MolecularGenetics (3rd ed) 853-1262 (1980) and Rose, et al., Manual of ClinicalImmunology (2d ed.) 612-713 (1980), which are incorporated herein byreference. Cultures of many of these viruses can be obtained from theAmerican Type Culture Collection (ATCC) in Rockville, Md.

When the appropriate ganglionic blocking agent is administered to amammal infected by one of these viruses, the treatment prevents ortreats infections or diseases caused by the virus. Additionally, theadministration of the ganglionic blocking agent can achieve one or moreother effects, including preventing or reducing the shedding of thevirus; preventing the recurrence of the disease caused by the virus inthe case of viruses causing recurring diseases; reducing the frequencyof the recurrence of symptoms of the disease in the case of virusescausing recurring diseases; reducing the duration of symptoms of thedisease; or reducing the severity of the symptoms.

In one embodiment, the ganglionic blocking agent is administered to themammal in a dosage effective to produce an anti-viral effect withrespect to the virus. In another embodiment, the ganglionic blockingagent is administered to the mammal in a dosage effective to inhibit theviral function of the virus. Of course, the same administration of theganglionic blocking agent to a mammal can achieve simultaneously many,if not all, of these effects.

One skilled in the art can select the appropriate ganglionic blockingagent and the appropriate dosage to produce these effects with respectto a particular infection or disease. For example, TEAC can beadministered to humans infected by HSV-1 or HSV-2 to achieve thesedesirable effects.

The ganglionic blocking agents can be administered to a mammal, such asman, in a number of pharmaceutically acceptable manners, such asintravenously, intramuscullarly, parenterally, topically, orally and/orvaginally. The particular ganglionic blocking agent to be selecteddepends on many factors including the virus implicated, the host, andthe proposed method of administration.

The ganglionic blocking agent will usually be present in apharmaceutically effective carrier or diluent, such as normal sterilesaline solution for systemic use or hydrophilic ointment bases fortopical or vaginal administration. The concentration of the ganglionicblocking agent is limited only by the amount that may be carried ordissolved in the carrier or diluent, but preferably is in the range fromabout 1 to about 1000 mg per ml (total volume), more preferably in arange of from about 50 to about 250 mg/ml. In an ointment theconcentration of the ganglionic blocking agent may range from 0.01% to99% of volume.

The dosages of the ganglionic blocking agent can be readily determinedby the skilled artisan. For example, about 200 mg to about 500 mg ofTEAC are intravenously administered to a human infected with HSV-1 orHSV-2, but not in an amount to exceed 7 mg per kg of body weight. Theintramuscular dosage of TEAC in adult humans is 1,000 mg to 1,200 mg,but not to exceed 20 mg per kg of body weight.

The ganglionic blocking agent can also be encapsulated or placed in apill form for oral administration to the host.

In one embodiment, the present invention provides a method for thetreatment or prevention of infections or diseases caused by HSV-1 andHSV-2. In such a treatment, ganglionic blockers, such as TEA ion orhomologs thereof are administered to an infected or diseased mammal toprevent or treat infections or diseases resulting from the HSV-1 andHSV-2.

Infection and disease by HSV-1 is typically associated with oral, facialand ocular lesions. Infection and disease by HSV-2 usually result ingenital and anal lesions.

Both HSV types 1 and 2 show a predilection for ectodermal tissues, asevidenced by their production of lesions in the skin, oral cavity,vagina, conjunctiva and the nervous system. HSV-2, which is usuallytransmitted venereally, is now epidemic in the United States. Sometwenty million persons are presently afflicted with this disease in thiscountry. New cases and recurrences exceed 500,000 annually. HSV-2infections often cause blindness, neonatal deaths, and encephalitis.

An important characteristic of HSV-1 and HSV-2 is their ability topersist in a latent or quiescent form in man and animals. Initial orprimary infections by HSV-1 and HSV-2 are contracted through breaks inthe mucous membrane in which the viruses replicate locally. From themembranes, the HSV-1 and HSV-2 spread to the regional lymph nodes and,occasionally, they can invade the bloodstream producing viremia. Whenthe primary infection subsides or recedes, the virus persists in alatent form in the sensory ganglia that innervate the site of primaryinfection. In ocular or oral infections, the HSV-1 and HSV-2 persist inthe trigeminal ganglia. In genital infections, the viruses persist inthe sacral ganglia.

Although the state of the viral genome during latency is not yet known,latency can be upset resulting in viral multiplication of HSV-1 andHSV-2. This multiplication produces the second or recurrent form of thedisease. Recurrences usually occur at the primary sites. In humans, suchrecurrent disease and infection are typically induced by heat, cold,sunlight, ultraviolet light, hormonal and emotional disturbances, orimmunosuppressive agents.

Epidemiological control of HSV-1 and HSV-2 is presently poor, becausethe majority of the population, up to 95%, has been exposed to theviruses. In the healthy carrier, the viruses can be isolated in variousfluids, such as tears, saliva, and vaginal secretions, even during theabsence of overt disease.

HSV-1 and HSV-2 have been experimentally produced in animals by avariety of stimuli, including physical manipulation of the sensoryganglia. In all cases of reactivation, a change is seen in thereservoirs of the ganglia. Therefore, the present inventor has foundthat ganglionic blocking agents that achieve ganglionic blockage in thehost have a profound effect on inhibiting the recurrence of infectionand diseases caused by HSV-1 and HSV-2.

The following examples demonstrate that ganglionic blocking agents areeffective in treating diseases and infections caused by viruses,especially HSV-1 and HSV-2.

EXAMPLE NO. 1

In the following experiments, the antiviral activity of two ganglionicblockers, tetraethylammonium chloride (TEAC), a TEA ion, andhexamethonium bromide (HMB), were tested. The tests showed that theseganglionic blockers inhibit the in vitro replication of HSV-1 and HSV-2.

The experiments in Example No. 1 were divided into three (3) series. Thefirst and second series of experiments in Example No. 1 evaluated theantiviral activity of tetraethylammonium chloride (TEAC), a ganglionicblocking agent that is also a monoquaternary ammonium compound. Theantiviral properties of TEAC were determined particularly with respectto the in vitro inhibition of HSV-1 and HSV-2 replication. The thirdseries of experiments in Example No. 1 determined the antiviral activityof hexamethonium bromide (HMB), a ganglionic blocking agent that is alsoa bis-quaternary ammonium compound.

In Series No. 1 of Example No. 1, monolayer cultures of Vero, RK-13(rabbit kidney), and WISH (human amnion, Hayflick) cells were used inthe experiments of Example No. 1. The cells were grown in Basal MinimalEagle's Media supplemented with 5% fetal calf serum, 1% glutamine,sodium bicarbonate and antibiotics. Cultures were maintained in amaintenance media that was the same media, except that the maintenancemedia contained 2% fetal calf serum. An F Strain of HSV-1 was chosenbecause it is a well-known prototype of HSV-1. This F Strain can beobtained from the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852, at which it is identified asATC-VR 733. For studies with HSV-2, a G strain of HSV-2 (ATCC-VR 734)and a 333 strain of HSV-2 were selected. These strains also can beobtained from the ATCC.

Two methods were used to test the drug's effectiveness in thesuppression of viral growth. In the pre-inoculation experiments, thedrug was added after adsorption of the virus. In the pre-incubationexperiments, the drug was added prior to infection of the cell cultures.

Accordingly, the pre-inoculation experiments measured the effect of thedrug on a cell that was already infected with the virus. Thepre-incubation experiments measured the cell cultures that were treatedwith the drug prior to the inoculation to determine if these cells thenwould be permissive or non-permissive to the tested virus. In theseexperiments, plaque reduction assays provided a quantitative measure ofantiviral activity and was deemed to be reliable and objective.

In the pre-inoculation experiments, monolayer cultures of WISH cellswere grown in either 25 cm² plastic flasks or 6 well cluster dishes andwere inoculated with 0.2 ml of the virus strain. The cells were allowedto adsorb the virus for 45 minutes at 37° C., with gentle rocking of thecultures every 15 minutes.

After the adsorption period, 1 ml of the drug and 3 ml of themaintenance media were added to each well. Also after the absorptionperiod, 1 ml of the drug and 4 ml of the maintenance media were added tothe 25 cm² plastic flasks. All cultures were prepared in duplicate.

In the pre-incubation experiments, the media was either decanted orgently aspirated from the cultures. After decanting or aspirating, 1 mlof the drug dilution plus maintenance media was added to the cultures.The cultures that served as virus controls or cell controls received 2ml of maintenance media. After the pre-treatment period, the media wasagain aspirated and the cell cultures were infected with the virus.

The cultures were incubated at 37° C. for 36-38 hours or until the viruscontrol showed discrete visible plaque. When this occurred, the cultureswere fixed and stained with crystal violet. The plaque were thencarefully counted under the microscope. By such a counting and carefulcomparison, percentages of plaque inhibition in the treated cultureswere then calculated.

To determine the toxicity level of the drug, all three celllines--RK-13, Vero cells, and WISH cells--were used. In the toxicityexperiments, the effect of the drug on the uninfected cell for a rangeof periods of time was determined. Accordingly, cultures were incubatedwith and without the drug. The time of exposure was from one to sevenhours. A comparison was then made between the drug tested cultures andthe control cultures. After each period of incubation, the cells werewashed and fresh media was added to the cells. The cell viability wasthen determined by dividing these cultures and having them grow into newcell cultures.

The apparent toxicity that was described as thin was totally reversible.When the drug was removed and fresh media added, the cell layer revertedto its normal non-treated appearance.

No change in morphology or cytopathic effect was seen in the culturescontaining TEAC nor those without TEAC. There was no difference in therate of growth between TEAC-tested cultures and cultures without TEAC.These observations indicate that TEAC is not toxic to cells.

All drug dilutions in the experiments were made in maintenance media.

These methods and materials were used in all of the experiments of thefirst series of Example No. 1.

A. FIRST SERIES EXPERIMENT 1A

The effect of TEAC on monolayer cultures of Vero and WISH cells wastested to determine the toxicity limits of the drug. Confluent monolayercultures of 25 cm² plastic bottles were incubated overnight atconcentrations of 5 mg, 2.5 mg and 1.25 mg of TEAC per culture. Nochange in the morphology of the cells was seen.

At the end of the incubation period, the cultures were washed twice withsterile PBS (Dulbecco). Fresh medium was added to each of the cultures.The cultures were then incubated for an additional 24 hours. There wasno apparent difference in the growth or general appearance between thetreated and the untreated controls.

EXPERIMENT 1B

Confluent monolayer cultures of Vero cells were infected with the Fstrain of HSV-1. After the adsorption of the virus by the cells, thecultures received 1 ml of TEAC at a concentration of 2.5 mg/ml plus 4 mlof maintenance media. The control cultures received no drug. Thecultures were stained at the appropriate time and the plaque werecounted.

Drug-treated cultures had an average of 75.5 plaque and the controlcultures had an average of 488.5 plaque. Therefore, the rate of plaqueinhibition was 84%. In addition, the size of the plaque was appreciablysmaller in the drug treated cultures as compared to the controlcultures.

EXPERIMENT 1C

Confluent monolayer cultures of WISH cells were infected with the Fstrain of HSV-1, as in Experiment 1B. The dilutions of TEAC tested were10 mg/ml, 5 mg/ml, 2.5 mg/ml and 0.65 mg/ml. After the adsorption of thevirus by the cells for 45 minutes, 1 ml of TEAC dilution plus 3 ml ofmaintenance media was placed into each of two wells.

In another plate, duplicate wells received 1 ml of TEAC dilution plus 1ml of maintenance media. These duplicate wells were incubated for 3hours. After this time, the maintenance media was aspirated and thecultures were infected. Fresh maintenance media was then added. Thecells were incubated at 37° C. until discrete plaque were observed inthe control wells.

The findings of experiment 1C are presented in Table 3. The findings canbe summarized as follows:

1. Antiviral activity was seen in all dilutions tested.

2. When the drug was added after inoculation with the virus, there wasno strict correlation between the viral inhibition and dosage. Forexample, TEAC concentrations of 5 and 2.5 mg/ml, which differ by afactor of 2, gave similar degrees of inhibition, namely, 45% and 46%.

3. Pre-incubation of the cell monolayer with 10 mg/ml of TEAC prior toinoculation with the virus gave 71% plaque inhibition. Pre-incubation ofthe cell monolayer with 5 mg and 2.5 mg of TEAC prior to inoculationgave 22% and 10% plaque inhibition.

                  TABLE 3                                                         ______________________________________                                        EFFECT OF TEAC ON HSV REPLICATION                                             (HSV-1 - F STRAIN)                                                            mg/culture   % plaque inhibition                                              ______________________________________                                        A. Drug added after inoculation with virus                                    10.00        56                                                               5.00         45                                                               2.50         46                                                               1.25         39                                                               0.62         30                                                               B. 3 hours pre-incubation                                                     10.0         71                                                               5.0          22                                                               2.5          10                                                               ______________________________________                                    

As seen in Table 3, pre-incubation of the cells with 10 mg/ml solutionof the drug rendered the cells non-permissive to the virus, as shown bythe 71% plaque inhibition. When the TEAC was added after inoculation ofthe culture with HSV-1, significantly different drug dilutions from 10mg/culture to 0.62 mg/culture yielded similar degrees of plaqueinhibition. Consequently, without being bound by theory, it is believedthat the mode of action of TEAC is other than by viral metabolicprocess.

EXPERIMENT 1D

Experiment 1D used the same method and materials as Experiment 1C exceptthat the TEAC drug concentrations were increased by a factor of ten. Theresults are presented in Table 4.

                  TABLE 4                                                         ______________________________________                                        EFFECT OF TEAC ON MSV REPLICATION                                             (HSV-1 - F SRAIN)                                                             mg/culture                                                                              % plaque inhibition A                                                                       % plaque inhibition B                                 ______________________________________                                        100.0     toxic         79                                                    50.0      100(thin)     82                                                    25.0      100(thin)     79                                                    12.5      62            80                                                    6.2       20            Not Done                                              ______________________________________                                    

A. Drug added after virus adsorption.

B. Cultures re-incubated with the drug for 3 hours prior to infection.

Thus, the results of Experiment 1D confirmed the findings of Experiment1C. Pre-incubation of the cells with several dilutions of the TEAC druggave comparable degrees of plaque inhibition. Those cultures thatreceived the drug prior to inoculation gave relatively the same degreeof protection (79%-82%), regardless of the TEAC concentration.

In the other cultures in which the drug was added after the adsorptionperiod, the following was found: 100 mg/culture was toxic and the cellslifted off the plate. At the next concentrations, 50 and 25 mg/culture,no plaque formed, but the cell layer was "thin." As used herein, theterm "thin" means that the cells were still attached to the container,but the monolayer was not confluent and there were spaces between theindividual cells. The 62% plaque inhibition observed in the culturescontaining 12.5 mg/culture of TEAC was consistent with the 56% plaqueinhibition at 10 mg/culture of TEAC in Experiment 1C.

EXPERIMENT 1E

In this experiment, the antiviral activity of TEAC was tested atdifferent concentrations of the drug and at different pre-incubationperiods using the above noted methods and materials.

WISH cells were pre-incubated with 100 mg, 50 mg, 25 mg, 12.5 mg and 6.2mg of TEAC per culture for 2, 4, and 7 hours prior to infection withHSV-1 (F strain). The results of experiment 1E are set out in Table 5.

                  TABLE 5                                                         ______________________________________                                        EFFECT OF TEAC ON HSV REPLICATION                                             (HSV-1 - F strain)                                                            Pre-incubation time                                                                          mg/culture                                                                             % plaque inhibition                                   ______________________________________                                        2 hours        100      68                                                    "              50       24                                                    "              25       17                                                    "              12.5     18                                                    "              6.25     0                                                     4 hours        100      100(thin)                                             "              50       63                                                    "              25       37.5                                                  "              12.5     less than 20                                          "              6.25     less than 20                                          7 hours        100      100(thin)                                             "              50       95.7                                                  "              25       96.5                                                  "              12.5     76                                                    "              6.25     50                                                    ______________________________________                                    

In these controlled experiments, antiviral activity was observed to bedependent on both concentration and duration of exposure to the TEAC.For example, the percent inhibition ranges between 17% and 96.5% at 25mg/culture.

EXPERIMENT 1F

The effect of TEAC was tested on cultures infected by HSV-2 inaccordance with the above noted method. The 333 strain, which can beobtained from ATCC, was used. Table 6 below shows the results of thisexperiment.

The percent inhibition of 56% with 50 mg/culture at a pre-incubationtime of 2 hours is consistent with the results found with HSV-1. Basedon the results in Table 6, HSV-2 (333 strain) appeared to be even moresensitive to TEAC than HSV-1. The G strain of HSV-2, obtained from ATCC,was also tested and gave similar results.

                  TABLE 6                                                         ______________________________________                                        EFFECT OF TEAC ON HSV REPLICATION                                             (HSV-2 - 333 STRAIN)                                                          Pre-incubation time                                                                          mg/culture                                                                             % plaque inhibition                                   ______________________________________                                        2 hours        50       56                                                    "              25       42                                                    "              12.5     25                                                    "              6.25     0                                                     ______________________________________                                    

In summary, Experiments 1A to 1F in the first series of Example No. 1showed that:

(a) TEAC inhibited the in vitro replication of HSV-1 and HSV-2.

(b) The antiviral effect of TEAC was present whether it was added beforeor after viral inoculation.

(c) When TEAC was added before viral inoculation, it rendered the cellsnon-permissive to the virus and no plaque formed. In these experiments,the antiviral activity was time and dosage dependent.

(d) Suppression of viral growth was evidenced not only by the reducednumber of plaque, but also by their reduced size.

(e) Without being bound by theory, the mode of action of TEAC does notappear to involve impairment of the cell metabolic process.

(f) The observed toxicity or changes in the cell's morphology wastotally reversible.

B. SECOND SERIES EXPERIMENT 1G

Confluent monolayer cultures of vero cells were infected with varyingamounts of HSV-1 (McKrae strain) and allowed to adsorb for 30 minutesprior to the addition of TEAC. All cultures were incubated at 37° C. ina 5% CO₂ humidified atmosphere. The cultures were stained with crystalviolet to aid in counting the plaque.

                  TABLE 7                                                         ______________________________________                                        DRUG RESPONSE CURVE                                                                  mg/ml No. of Plaques                                                   ______________________________________                                               50    Cells lifted off                                                        25    0                                                                       12.5  300                                                                     6.25  286                                                                     3.125 247                                                                     VC    270                                                                     VC    270                                                                     VC    252                                                              ______________________________________                                    

As shown, the highest concentration (50 mg/ml) was toxic and the cellslifted off and died. The second concentration (25 mg/ml) was tolerated,but the monolayer had a thinned out appearance. The rest of theconcentrations was not significantly effective in achieving plaqueinhibition.

The data in Table 7 suggest the apparent enhancement of plaque found inthe cultures that received 12.5 mg/ml of the drug. Investigations werethen made of other concentrations that would give good antiviralactivity but less toxicity and a working concentration of 20 mg/ml ofTEAC was determined.

EXPERIMENT 1H

To determine whether TEAC physically inactivates the virus prior toentry into the cell, experiment 1H was conducted. An aliquot of a virussuspension was mixed with TEAC to yield a final concentration of 20mg/ml. A similar aliquot without the drug, but in the same volume, wasprepared as a control. Both mixtures were maintained at 4° C. for 30minutes, 60 minutes, or 90 minutes. 0.3 ml of the solutions were platedonto susceptible cell cultures and incubated at 37° C. for 36 hours. Allcultures were stained and compared to the virus control.

There was no discernible difference between the control group and theTEAC-treated group. This indicated that incubation of the cell-freevirus with TEAC does not inactivate the virus. The results aresummarized in Table 8.

                  TABLE 8                                                         ______________________________________                                        EFFECT OF TEAC ON CELL FREE VIRUS                                             Time           Drug-treated                                                                            Control                                              ______________________________________                                        30 min.        265       273                                                  60 min.        260       272                                                  90 min.        258       274                                                  ______________________________________                                    

EXPERIMENT 1I

Previously, cell toxicity at higher concentrations of TEAC in the rangeof 50 and 25 mg/ml was reported. To investigate the degree of toxicityin other cells, TEAC and Hexamethonium Bromide (HMB) at 25 mg/ml weretested. Healthy confluent monolayer cell cultures were overlaid with 1ml of the drug and incubated for 48 hours at 37° C. and then stained.Cell cultures with maintenance media were used as controls. The resultswere as follows:

(a) RK-13 cells showed toxicity with TEAC and slight toxicity with HMB;

(b) Flow 1000 cells (foreskin cells) showed toxicity with TEAC and notoxicity with HMB; and

(c) Vero cells showed slight toxicity with TEAC and no toxicity withHMB.

Consequently, it appeared that the toxicity was host cell dependent.

EXPERIMENT 1J

Large number of cultures were infected with HSV-1. After virusadsorption, maintenance media containing 20 mg/ml of TEAC was added. Atvarying times, the TEAC was removed and fresh media without TEAC wasadded to the cultures.

                  TABLE 9                                                         ______________________________________                                        ANTIVIRAL ACTIVITY OF TEAC                                                    TEAC removed    PFU/ml  % Reduction                                           ______________________________________                                        4 hours P.I.    1042    25%                                                   6 hours P.I.    596     57%                                                   8 hours P.I.    436     69%                                                   10 hours P.I.   163     88%                                                   Virus Control   1398                                                          ______________________________________                                    

Therefore, a significant reduction in plaque was achieved at 10 hoursP.I. as shown by 88% plaque inhibition.

EXPERIMENT 1K

This experiment attempted to measure the antiviral activity of TEACexpressed as total virus yield. In experiment 1K, cultures were infectedwith the McKrae strain of HSV-1 at a high multiplicity of infection. At4 hours post infection, when viral replication was underway, TEAC at aconcentration of 20 mg/ml was added to the cultures. At designatedtimes, the cultures were harvested. The supernatant fluid and the cellswere kept separate to assay for viruses released in the supernatantfluid and also that present in the cells. Both supernatant fluid and thecells were frozen immediately at -85° C. These samples were then assayedfor infectious particles on susceptible cells. The results are shown inTable 10.

                  TABLE 10                                                        ______________________________________                                        VIRUS YIELD FROM TREATED AND NON-TREATED                                      RE INFECTED CELLS IN PFU/ml                                                   ______________________________________                                        SUPERNATANT                                                                   TEAC                 Control                                                  Time P.I.                                                                             10.sup.-1                                                                             10.sup.-2                                                                              10.sup.-3                                                                           10.sup.-1                                                                           10.sup.-2                                                                           10.sup.-3                          ______________________________________                                        4 hours N.D.    N.D.     N.D.  2     0     0                                  6 hours 0       0        0     3     1     0                                  10 hours                                                                              0       0        0     22    7     0                                  20 hours                                                                              0       0        0     91    10    0                                  24 hours                                                                              0       0        0     107   18    2                                  28 hours                                                                              0       0        0     190   58    7                                  ______________________________________                                        CELL                                                                          TEAC                 Control                                                  Time P.I.                                                                             Und.    10.sup.-1                                                                              10.sup.-2                                                                           Und.  10.sup.-1                                                                           10.sup.-2                          ______________________________________                                        4 hours N.D.    N.D.     N.D.  20    9     4                                  6 hours 12      2        5     16    7     7                                  10 hours                                                                              13      3        0     48    12    2                                  20 hours                                                                              42      5        0     W.O.  W.O.  W.O.                               24 hours                                                                              58      32       10    W.O.  W.O.  W.O.                               28 hours                                                                              W.O.    TMTC     15    W.O.  W.O.  W.O.                               ______________________________________                                         W.O. = wiped out                                                              TMTC -- too many to count                                                

EXPERIMENT 1L

Monolayer cultures of Vero cells were infected with the McKrae strain ofHSV-1. After the virus adsorption, the cultures were divided into twosets. One set received maintenance media with 20 mg/ml TEAC and theother set received the same media, but without the TEAC. At 30 min., 6hr. and 24 hr. post infection, both experimental and control cultureswere harvested. The cells were gently scraped and centrifuged.

The pellet was cut into 3 pieces that were double fixed in 2.5%glutaraldehyde and 1% osmium tetraoxide. The cells were dehydrated in aseries of increasing alcohol content solutions and embedded in Epon 812.One block of each series was used to cut thick sections for lightmicroscopy. Sections one micron thick from each block were stained withtoluidine blue and examined under a light microscope to select an areafor electron microscope (EM) observation. The selected area containednormal and infected cells in comparative amounts. The percentage ofcells exhibiting cytopathic effects is shown in Table 11.

                  TABLE 11                                                        ______________________________________                                        PERCENTAGE OF CELLS EXHIBITING CPE                                            AREA OF INFECTION PER FIELD                                                   Time P.I.    Virus Control                                                                            Virus and Drug                                        ______________________________________                                        30 mins.     scattered  N.D.                                                  6 hours      1/4        1/6                                                   30 hours     1/2        scattered                                             ______________________________________                                         N.D. = not detectable                                                    

Both virus control and drug-treated cultures, at 6 hours post infection,had formed dense intranuclear bodies. In the control group, the densebodies were from 43 to 60 A in size. At 6 hours post infection, theintranuclear bodies were more numerous and ranged in size from 43 to 70A. The drug-treated cells at 6 post infection also showed the formationof intranuclear bodies and the cell membrane was unusually extended andwavy. There was no striking difference in the number of cells containingintranuclear dense bodies in the control cells and the drug-treatedcells at 6 hours post infection. However, the extended and wavyappearance of the cytoplasma membrane in the drug-treated cultures wasof interest.

At 24 hours post infection, the control group showed nucleocapsids inthe nuclei of the cells and the virus particles budding from the cellmembrane. Numerous complete virions in the extracellular space were alsoobserved.

In contrast, the TEAC-treated cells at 24 hours post infection showedsome nucleocapsids of about 99.6/A in size and several virions withcoil-like cores in vacuoles. In the inter-cellular space, some abnormalvirus particles were detected and the cytoplasma membrane in thesecultures continued to be extended and wavy. Some of these TEAC-treatedcells had coreless nucleocapsids in paracrystalline arrays in theircytoplasma. The relative number of cells exhibiting these changes isillustrated in Table 12 below.

                  TABLE 12                                                        ______________________________________                                        EM EXAMINATION OF TREATED AND NON-TREATED CELLS                                       No. of Cells                                                                             Internuclear                                                                              Virus                                          Time P.I.                                                                             Examined   Dense Bodies                                                                              Particles                                                                             Total %                                ______________________________________                                        CONTROL                                                                       30 min. 100         6          0        6%                                    6 hours 100        14          0       14%                                    24 hours                                                                               50         0          50      100%                                   TEAC TREATED                                                                  30 min. N.D.       N.D.        N.D.    --                                     6 hours 100        12          0       12%                                    24 hours                                                                              300        12          8        6%                                    ______________________________________                                    

These observations indicate that the antiviral activity of TEAC may bedue to an impairment or block in the assembly of the viral componentsbut not in their production.

C. THIRD SERIES

In the experiments of the third series, it was investigated whether theantiviral activity of TEAC was shared by all ganglionic blocking agents.Confluent monolayer cultures of Vero cells were infected with theappropriate amount of HSV-2. The 333 strain of HSV-2, which can beobtained from ATCC, was used and allowed to adsorb for 30 minutes priorto the addition of the drug.

EXPERIMENT 1M

In this experiment, 25 mg/ml HMB was tested on Vero cells infected withHSV-2. The virus control cultures, without HMB, had 705 plaque. TheHMB-treated cultures had 142 plaque and a thin cell layer. Consequently,80% plaque inhibition was achieved in the cell cultures treated withHMB.

EXPERIMENT 1N

In this experiment, 20 mg/ml of HMB was tested on Vero cells infectedwith HSV-2, 20 mg/ml of HMB gave complete inhibition of plaque. Thenumber of plaque on the virus control was about 10 to 12 plaque.

EXPERIMENT 1O

In this experiment, 25 mg/ml, 20 mg/ml, and 15 mg/ml of HMBconcentrations were tested on Vero cells infected with HSV-2. Theresults were: 15 mg/ml did not have any effect. The experimental flaskshad as many, or more, plaque as the control, with an average of 154plaque for the control and 169 plaque for the HMB-treated flasks.

At 20 mg/ml HMB, there was no plaque.

At 25 mg/ml HMB, the HMB was toxic to the monolayer. Hence, althoughthere were no plaque, the lack of plaque was most likely due to toxicityrather than antiviral activity. In this experiment, the bottles werestained at a later time than in other experiments because the controlgrew slowly.

EXPERIMENT 1P

HMB was further tested at a concentration of 20 mg/ml of re infectedVero cells. The control flasks had an average of 89 plaque and theHMB-treated flasks had an average of 7 to 8 plaque. This represented aplaque inhibition of 91%.

EXAMPLE NO. 2

In this experiment, TEAC was tested against a non-herpeto virus todetermine whether its antiviral activity (and, therefore, that ofganglionic blocking agents as a group) was HSV specific or whether theantiviral activity extended to viruses as a whole. For this purpose, thevirus vaccinia was selected.

Confluent monolayer cell cultures of Vero cells were infected with the333 strain of vaccinia. After adsorption of the virus, maintenance mediacontaining TEAC was added to the flasks. The same maintenance mediawithout the TEAC was added to the control flasks. The flasks weretreated with TEAC concentrations of 25 mg/ml, 20 mg/ml, 15 mg/ml, and 10mg/ml. After 36-48 hours incubation, the plates were stained and theplaque were counted.

The flasks containing 25 mg/ml, 20 mg/ml, and 15 mg/ml of TEAC did nothave any plaque, but the layers were also noted to be thin. The flaskscontaining 10 mg/ml of TEAC exhibited only 2 or 3 plaque per flaskvisible under the microscope. Hence, at 10 mg/ml of TEAC, 100%inhibition of viral growth of the vaccinia occurred. The control flasks,in contrast, exhibited well defined plaque averaging 150-160 plaque.

EXAMPLE NO. 3

In Vivo Experiments

Female guinea pigs (obtained from Charles River, Inc.) weighingapproximately 200 g were used as a model for HSV-2 skin lesions. Theanimals were anesthetized with 0.15 ml of Ketamine-Rompum mixture. Theanimals were first shaved on their back with a hair clipper and thentreated with a depilatory cream (Nair). The denuded area was washed welland then dried. A tuberculin tyne was washed with ethanol and air dried.

Two methods were tried for infecting the animals with the virus. In one,a drop of HSV-2 suspension was placed on the skin and the skin waspunctured with the tyne. In the second method, the tyne was dipped inthe virus suspension before puncturing the skin. The first method gavebetter results and, therefore, was adopted.

Lesions were evident 4 days after infection.

The vesicles had an erythematous base and they were elevated with ablistering appearance. When the vesicles ruptured, a crust formed. Thelesions resolved by day 9 or 10 post infection.

In the first experiment, each of 3 guinea pigs was infected on threeareas of their backs. At 3 days post infection, treatment was begun onone side of each animal, leaving the other side untreated as a control.

The TEAC treatment was 4 times daily. The animals were examined dailyfor vesicles (V), crust (C) and for erythema (R) alone. Lesions on thedrug-treated areas did not crust but appeared to be faded by 6 days postinfection (pi) or day 3 after treatment. At day 4 after treatment, arash appeared. Since it was present in any other area, this dermatitiswas probably due to the TEAC or its vehicle or both.

In the next experiment 6 guinea pigs were infected in the same manner asthe initial 3 guinea pigs. Treatment was begun 12-16 hours postinfection. Three animals were treated with TEAC ointment, while theother three served as controls. Lesions showing an erythematous basewith either a blister or a crust on top were considered to HSV specific.These criteria were used because dermatitis made readings difficult.Both the control animals and treated animals were carefully read in thesame manner. There was a marked difference between the two groups, notonly in the number of lesions but also in their appearance. The resultsrelating to the number of lesions are provided in Table 13.

                  TABLE 13                                                        ______________________________________                                        REDUCTION OF LESIONS BY TEAC                                                  # of lesions                                                                  Days P.I.                                                                              Control    TEAC Treated                                                                             Percent Reduction                              ______________________________________                                        3        19         15         21                                             4        40         29         27.5                                           5        41         17         58.5                                           6        36         15         58.3                                           ______________________________________                                    

Thus, regardless of whether the animals were treated 3 pi or treated12-16 hours pi, the TEAC was very effective in treating or preventingthe HSV-2. When given 3 days pi, the vesicles die not crust; rather,they reduced in elevation and eventually resolved. When the TEAC wasgiven early, 12-16 hours post infection, less vesicles formed. Not onlywas there a significant reduction in the number of lesions, but also intheir size. Moreover, the lesions neither ruptured nor crusted. As isevident from the experiments, the TEAC had a significant effect inreducing the number and size of HSV-2 lesions, as well as inhibiting therupture and crusting up the HSV-2 lesions.

EXAMPLE NO. 4

Clinical Tests

Clinical Case A

Patient A is a 43 year old female who first report with a history ofherpetic lesions on her buttocks which had erupted approximately every30 days for several years previously (over a 10 year period, withincreasing frequency during the past 2 years). She was referred by aboard certified dermatologist who had diagnosed HSV type 2 and who hadtreated the lesions of at least five attacks with steroid injections.The lesions usually lasted from between seven days to six weeks. Thepatient experienced periodic pain in the buttocks with chronic severesciatic pain radiating down her right leg.

Patient A appeared for treatment two days following the latest eruptionof lesions on her buttocks. She weighed 57 kg. and was administered 2.8cc. of a solution of 100 mg of TEAC per cc. of normal saline. (Thissolution was used in all parenteral administrations described herein.)At the time of treatment she experienced some blurred vision and slightdizziness on rising to the seated position. However, immediatelyfollowing the infusion she reported cessation of sciatic pain.

The patient called approximately three weeks later reporting that shewas asymptomatic. She subsequently reported that she had gone 52-53 dayswithout lesions or pain at which time a few vesicles had appearedwithout weeping or crusting and disappeared in two to three days withoutpain.

Two months after the initial exam, the patient reported that severalsmall lesions had developed on the previous day with mild sciatic pain.She was given a second infusion of 3.2 cc. Immediately after thistreatment she experienced some blurred vision but the sciatic nerve paindisappeared during the infusion.

Three days later, the patient had 6 to 8 patches of lesions developingover a larger area with some pain but no vesicles had developed yet. Thepatient also had pain in the back and down both legs.

Two days later, the patient was given a third infusion of 3.4 cc.

Approximately two weeks later, the patient reported a few tiny areasunder the skin but without eruptions and the tiny areas disappeared inabout a day. About three weeks later there was no further pain orlesions. About three weeks later, the patient continued to be troublefree. About one week later the patient reported that a few spots came upwithout aura, and that these were gone in 24 hours.

The patient was last contacted eighteen months after the initial exam atwhich time she advised that she had experienced no further lesions orpain.

Clinical Case B

Patient B is a 43 year old male diagnosed as having both HSV type 1(expressed as oral lesions) and HSV type 2 (expressed as penilelesions). He weighed 75 kg when he reported and was administered 3.7 ccof TEAC (5 mg/kg). Immediately following infusion, the patient reportedblurred vision, dryness of the mouth, and unsteady gait; however, thesedrug related effects ceased 10-15 min. after cessation of infusion.

In a call to the patient 3 days later he reported that his oral lesionshad been resolved, resolution occurring in a shorter than usual time.

About three weeks later, the patient described a labial aura andpredicted a massive outbreak. He was administered an additional 3.7 cc.The following day the patient reported an absence of an aura and nooccurrence of the expected lesion except for a very small lesion at thecorner of the mouth which was cultured out as HSV-1. About a month laterthe patient reported a "chapped lip-like" area but that the attack wasnever serious and that no lesion appeared.

Five weeks after the previous treatment, the patient returned fortreatment of acute penile lesions of HSV type 1 and 2. He wasadministered 4.5 cc (6.0 kg/mg). Eight days later, the patient reportedthat the lesion had dried the next day and crusted and disappeared in2-3 days. The patient reported feeling that his active lesions obviouslyresponded to treatment but that recurrences were not eliminated.

About three days later, lip lesions appeared, and the patient requestedfurther treatment. He was administered 4.8 cc of TEAC. About 5 dayslater, the patient reported that labial and penile lesions werestarting. The patient was contacted about two weeks later, and reportedthat the last treatment appeared of no value. He reported that he hadhad painful penile lesions that lasted 8 days. About a month later,however, the patient reported that he had no more attacks, which heattributed in part to the treatment.

Clinical Case C

Patient C is a 26 year old male who weighed 77 kg when he reported fortreatment. He was diagnosed as having multiple herpes simplex virusinfection since the age of 6 months. He appeared for treatment withlabial lesions and a large lesion in the middle of his back. He also hadlesions on his forehead. In the past he had lesions nasally. He had attimes experienced remissions of 4-5 months.

The patient was infused with 3.85 cc (5 mg/kg) TEAC solution, andexperience slightly blurred vision.

The next day, the patient reported that the lesions were markedlyimproved, especially on his forehead. Three days later, the patientexperienced lesions. However, three additional days later it wasacknowledged that two attacks of labial lesions cleared rapidly. A weeklater, the patient reported having only a few fever blisters thatcleared right away. The patient reported the same a week later. Noproblems were reported until about a month later when the patientreported a lesion on the forehead that crusted relatively quickly. Thepatient was contacted five months and ten months after the initialtreatment reported that he had experienced no further problems.

Clinical Case D

Patient D is a 37 year old female who weighed 56 kg when she reportedfor treatment. For the past 5 years, she had experienced lesions on herbuttocks, with symptoms worsening prior to her menstrual cycle. Therewas no time she reported when there was a cessation of lesions or auras.

She was administered 2.8 cc TEAC solution (5 mg/kg) and experienced drymouth and transient blurred vision. Two days later she reported that alllesions were improving and that itching and tingling disappeared withthe treatment. She has not been available for further follow up.

Clinical Case E

Patient E is a 34 year old male who weighed 93 kg when he reported fortreatment. He had a history of HSV type 2 penile lesions occurring atleast once per month with the lesions sometimes lasting 3 months. He wasadministered 4.8 cc of TEAC (5 mg/kg), and experienced slightly blurredvision. Two days later, he reported that the lesions had started tocrust; however, he then experienced a massive prodome with large weepinglesions which were unlike any lesions of the previous 5 years.

The patient returned two weeks later without lesions but feeling that hewas in a "prodome stage". He was administered another 4.8 cc of TEACsolution on that day. The next day he reported no prodome and nolesions, but the following day reported that another breakout wasoccurring.

A week later, the patient was 99% cleared with 2 pinpoint lesionsremaining. He was infused with a further 5 cc of TEAC on that day. Threedays later the patient reported only pinpoint sites and felt that eachinfusion caused a definite change in the disease. Two days later thepatient reported that all lesions had cleared up. A few days later,vesicles had appeared which quickly disappeared without crusting, andone new lesion had appeared.

Three days later, the patient reported that the final lesion had gonebut that "some lesions are trying to come". Two days later the lesionshad not come. A week later, the patient reported that prodome started 3days earlier with 5-6 pinpoint lesions breaking out on the following dayon the right dorsal side of his penis.

Clinical Case F

Patient F is a 31 year old male diagnosed as having HSV type 2 infectionof his penis. He reported a history of penile lesions approximatelyevery two months lasting 1-2 weeks. He weighed 75 kg when he reported,and was administered 3.8 cc (5 kg/mg) of TEAC. The next day the patientconsidered his lesion improved. The following day the patient'sdermatologist found a small lesion. Three weeks later the patientnoticed a small new lesion.

A month after initial treatment, the patient reported with a lesion onthe base of his penis and was administered 4.5 cc of TEAC. Nine dayslater the patient stated that the lesion had cleared in 48 hours, but onthe other side lesions had developed.

The next day the patient was infused with a further 4.0 cc of TEAC.Within 4 days the lesions had cleared up. About 3 weeks later, thepatient reported that a new lesion had appeared earlier at the samesite, was open for a day or so, and then crusted and that now hisgeneral condition was the best it had been for several months and thathe was completely cleared up.

Five weeks later, the patient reported that he had had one lesion thatlasted a week and another that cleared up quickly and that he was in"good shape". The patient subsequently had lesions that lasted 12 daysand 7 days.

Clinical Case G

Patient G is a 36 year old male who weighed 78 kg and was diagnosed ashaving HSV type 2. He reported that penile lesions had erupted every 2weeks to 3 months (usually every 1-11/2 months) for the previous 7years. Lesions were acute for 2-3 days followed by 4 days of scarring.

When he reported for treatment, the patient reported experiencinglabilais prodrome without lesions and also a sensation in his lowerback. He was administered 4.3 cc of TEAC solution on that day. The nextday he reported that the treatment to be "great" with the lesionsimproving, and 2 days later he reported the "lesions" were gone with nocrusting, skin smooth, visable capillaries disappeared, softercircumcision scar, no back sensation, psoriasis improved, but somereddening of the lip where the patient had never had a lesion. 3 dayslater he reported everything fine except two pinpoints red spots thatthe patient did not feel would become vesicles. A week later, thepatient reported that the lesions had never crusted and were gone within48 hours.

After over a month with only a minor tingling incident the patientreported 2 penile lesions and was administered 4.5 cc of TEAC solution.He was also infused with a further 4.5 cc of TEAC the next day. Thepatient remained relatively stable for about a month; however, after anadditional month with no new infusion, the patient was back to hispre-treatment state.

The patient was contacted five months after initial treatment at whichtime he stated that he was at his pre-treatment state.

Clinical Case H

Patient H is a 41 year old male who weighed 77 kg when he reported. Hewas diagnosed as having HSV type 2. The patient reported that for thelast 2 years he had had penile lesions (lasting 7-10 days) approximatelyevery 2 months. A lesion had started the day before he reported. He wastreated with 4.6 cc of TEAC (6.0 mg/kg). The following day the patientreported striking improvement, but experienced a setback 6 days laterwhen the condition became a visual attack. The attack disappeared.

The patient was contacted six months after treatment, at which time headvised that besides 2 minor incidents of red spots (which did notdevelop) and some aura, he has had no further problems and, inparticular, no further vesicles since the attack immediately followinghis treatment cleared.

Clinical Case I

Patient I is a 42 year old male who weighed 86 kg when he reported fortreatment. He was diagnosed as having both HSV types 1 and 2. Thepatient reported he had had good health until he developed herpeticlesions in and around the mouth, chin and cheeks, unexplained nocturnalheadaches, penile lesions and symptoms similar to those of stomachulcers. He stated his lesions were almost constantly present. He wasinfused with 4.7 cc of TEAC solution. The next day, the patient reportedthat a sore had started on his tongue, but had gone by that morning.Four days later the patient reported that the infection tried to come upall over but failed and he was all clear.

About 5 weeks later, the patient reported with a lesion on his chinwhich had developed the previous day. He also reported a nocturnalheadache the previous night. He stated that these were the firstproblems he had had since his treatment. He was administered a further4.75 cc TEAC solution (5.5 mg/kg body weight).

Three months later the patient reported that the lesion had cleared 3days after the infusion and that he had had no further lesions until onedeveloped in the preceding week. He reported that he had had no furthernocturnal headaches.

Clinical Case J

Patient J is a 50 year old female who weighed 66 kg when she reportedfor treatment. She was diagnosed as having HSV type 2. She described ahistory of sacral lesions during the past 4 years which occurred every2-3 months. Lesion lasted for 5-30 days. She reported moderate pain,itching and stinging in the sacral region commencing the previous night.

She was infused with 3.6 cc of TEAC (5.5 mg/kg) and experienced someblurred vision immediately following the treatment.

Two days later the patient reported that the pain had disappeared andthat the lesions were drying. Five years later the patient reported thatblisters had appeared two days previously but had now cleared. Twomonths after treatment the patient reported tingling blisters a monthlater she was given TEAC in a 1% concentration in a hydrophilic ointmentbase for topical administration. The following day she reported that thetingling and redness had gone but that 2 more vesicles had appeared.Three days later the patient reported that all the lesions had dried up.

The patient was again contacted six weeks later when she reported thatshe had since had one attack but that the lesion had only lasted 3 days.On that occasion the patient had once again applied the ointment. Thispatient was contacted 12 months later at which time she advised that shehas had very few incidents since treatment, but that she has appliedointment and the problem has resolved itself completely.

Clinical Case K

Patient K is a 34 year old female who weighed 59 kg. when she reportedfor treatment. She was diagnosed as having HSV type 2. She described ahistory of lesions on the buttocks and genital region with attacksoccurring monthly in cooler weather and weekly in warmer weather. Shesaid her last attack had occurred one week prior to her reporting fortreatment. The patient was infused with 3.1 cc, after which sheexperienced some blurred vision. Three weeks later the patient reportedthat she felt a massive attack coming. On the same day the patient wasadministered a further 3.4 cc. of TEAC after which she once againexperienced blurred vision. Six weeks later the patient reported thatthe attack had not occurred. She said that she had since had 2 shorterattacks (of about 2-3 days each) both of which were of a milderseverity.

Clinical Case L

Patient L is a 49 year old female who weighed 53 kg. when she reportedfor treatment. She was referred by a Board Certified Dermatologist whohad performed facial dermabrasion on her. She had developed HSV Type 1lesions throughout the area of the surgery. After the development of thesurgical complication, the patient could recall one previous lesion in alocalized area some years previously.

The patient was infused with 2.9 cc. of TEAC (5.5 mg/kg) after which sheexperienced some blurred vision. Four days after treatment the patientreported that she had felt badly for 24-36 hours following the treatmentbut had since felt quite well.

Three weeks later the patient's referring dermatologist advised that onthe 7th day after the treatment, the crusting had all disappeared. Heobserved that in his experience this would normally have taken 3 weeksuntil DMSO treatment was introduced after which time he had still notseen it take less that 2 weeks.

Clinical Case M

Patient M is a 22 year old female with a history of HSV type 1infections extending over a period of more than 2 years. When shereported for treatment, she described a history of labialis lesionsoccurring every 2-3 months and lasting approximately 14 days. Thelesions would typically begin as blisters then spread out to a moreextensive area. When the patient reported, a lesion had begun 21/2 dayspreviously. She was given an ointment containing a 1% concentration ofTEAC in a hydrophillic ointment base for topical administration. Fivedays later, the patient reported that the lesions had cleared in ashorter time period than usual.

Two months later, the patient once again reported that a labialis lesionhad developed and that she had once again resumed administration of theointment. Three days later, the patient reported that the lesion had notspread and had now crusted.

CLINICAL CONCLUSIONS

From the above clinical cases, it is clearly seen that administration ofganglionic blockers was effective in the treatment of HSV types 1 and 2diseases. The degree of relief and the duration of the relief variedfrom patient to patient, ranging from a minor short-term effect tocomplete remission of the disease. In some cases, no significantimprovement was reported. Thus, while treatment with a ganglionicblocker is not the to all HSV problems, it can provide very significantand welcome relief for those patients that do respond.

CONCLUSIONS

In the in vitro and clinical experiments, two ganglionic blockingagents, TEAC and HMB, and three viruses, HSV-1, HSV-2, and vaccinia,were selected and tested as representative of ganglionic blocking agentsand viruses. The experiments clearly establish that ganglionic blockingagents are effective in treating and preventing diseases and infectionscaused by viruses.

It is intended that the specification and examples be considered asexemplary only with the true scope and spirit of the invention beingindicated by the claims.

Because many varying and different embodiments may be made within thescope of the inventive concept herein taught, and because manymodifications may be made in the embodiments herein detailed inaccordance with the descriptive requirements of the law, it is to beunderstood that the details herein are to be interpreted as illustrativeand not is a limiting sense.

What is claimed is:
 1. A method of treating a mammalian disease causedby a DNA virus comprising the step of administering to the mammal aganglionic blocking agent selected from the group consisting oftetraethylammonium ion, hexamethonium ion, pentolinium ion,chlorisondamine ion, trimethidinium ion, trimethaphan ion, mecamylamine,pempidine, and homologs thereof in an effective dosage, with the provisothat when the DNA virus is herpes zoster the ganglionic blocking agentis not tetraethylammonium chloride.
 2. The method of claim 1, whereinthe ganglionic blocking agent is selected from the group consisting oftetraethyl ammonium ion, hexamethonium ion, and homologs thereof.
 3. Themethod of claim 1, wherein the ganglionic blocking agent is administeredin a pharmaceutically acceptable carrier.
 4. A method of treating amammalian disease caused by a DNA virus comprising the step ofadministering to the mammal a ganglionic blocking agent selected fromthe group consisting of tetraethylammonium ion, hexamethonium ion,pentolinium ion, chlorisondamine ion, trimethidinium ion, trimethaphanion, mecamylamine, pempidine, and homologs thereof in a dosage effectiveto produce an anti-viral effect with respect to a DNA virus, with theproviso that when the DNA virus is herpes zoster the ganglionic blockingagent is not tetraethylammonium chloride.
 5. The method of claim 4,wherein the ganglionic blocking agent is selected from the groupconsisting of tetraethyl ammonium ion, hexamethonium ion, and homologsthereof.
 6. The method of claim 4, wherein the ganglionic blocking agentis administered in a pharmaceutically acceptable carrier.
 7. A method oftreating a mammalian disease caused by a DNA virus comprising the stepof administering to the mammal a ganglionic blocking agent selected fromthe group consisting of tetraethylammonium ion, hexamethonium ion,pentolinium ion, chlorisondamine ion, trimethidinium ion, trimethaphanion, mecamylamine, pempidine, and homologs thereof in a dosage effectiveto inhibit the viral function of a DNA virus, with the proviso that whenthe DNA virus is herpes zoster the ganglionic blocking agent is nottetraethylammonium chloride.
 8. The method of claim 7, wherein theganglionic blocking agent is selected from the group consisting oftetraethylammonium ion, hexamethonium ion, and homologs thereof.
 9. Themethod of claim 7, wherein the ganglionic blocking agent is administeredin a pharmaceutically acceptable carrier.
 10. A method of treating amammalian disease caused by a Papovavirus, comprising administering tothe mammal a ganglionic blocking agent selected from the groupconsisting of tetraethylammonium ion, hexamethonium ion, pentoliniumion, chlorisondamine ion, trimethidinium ion, trimethaphan ion,mecamylamine, pempidine, and homologs thereof in an effective dosage.11. A method of claim 10, wherein the ganglionic blocking agent is aquaternary ammonium compound.
 12. The method of claim 10, wherein thePapovavirus is a papilloma virus.
 13. The method of claim 10, whereinthe ganglionic blocking agent is a tetraethylammonium ion or a homologthereof.
 14. The method of claim 13, wherein the Papovavirus is apapilloma virus.
 15. A method of treating a mammalian disease caused bya Cytomegalovirus, comprising administering to the mammal a ganglionicblocking agent selected from the group consisting of tetraethylammoniumion, hexamethonium ion, pentolinium ion, chlorisondamine ion,trimethidinium ion, trimethaphan ion, mecamylamine, pempidine, andhomologs thereof in an effective dosage.
 16. A method of claim 15,wherein the ganglionic blocking agent is a quaternary ammonium compound.17. The method of claim 15, wherein the ganglionic blocking agent is atetraethylammonium ion or a homolog thereof.